Introduction
The pGem-gbr22 protein was expressed, purified and characterized. Recombinant proteins for expression in E. coli is used in biomedical science and E. coli are used as an expression host for proteins from higher organisms to distinguish between the protein and everything else[1]. More specifically, BL21(DE3) is an excellent host strain with deficiency in both lon and ompT proteases, which can enhance accumulation by reducing proteolytic degradation. [2]. The main idea of this lab of expression, purification and characterization, was to transform competent cells and grow cultures to express the colored (purple) protein and harvest cells by using the centrifuge. Another reason was to express and analyze purified gbr22 protein initially grown in bacteria using gel electrophoresis and UV- Vis spectroscopy. After lysing the cell, centrifuging, and using Ni-NTA affinity purification, the gbr22 protein should only remain in the sample. Materials and methods
Appropriate safety precautions such as gloves, glasses and lab coats to protect against stains, bacteria and chemicals were taken. Also, understanding the labs before beginning them was also a safety precaution. In order to start the first of the three procedures, cloning of the gene and inserting the gene into an expression plasmid was the first steps in expressing the protein. To begin, 25ul of the bacteria was added to each of the two transformation tubes and spun down using a mini centrifuge. 1-2ul of plasmid, which was pGEM-gbr22, was gently added to the bacterial in the DNA tube only and placed it on ice to prepare for the heat shock. After the shock, the samples were placed in ice for another two minutes. 200ul of SOC was added and placed in the incubator. Sterile colirollers were added to each DNA and Control plate to allow bacteria growth on the agar amp plates. The next day, ampicillin was added to the starter culture. A few colonies of the bacteria grown overnight were added to the LB. A few hours later, LB and ampicillin were added to a flask along with BL21(DE2) and then left on the shaking incubator. 8 hours later, the starter culture was transferred to a 125ml flask and put in the shaking incubator for 24 hours. On day three, the bacteria was placed in conical tubes and centrifuged for 10 minutes at 5,000 rpm at 4C. The pellet in the bottom was saved. 1xPBS was added to the cell pellet and vortexed in order to resuspend the cells in Phosphate buffered saline followed by lysozyme. Lysate from above procedure was distributed into several micro centrifuge tubes and centrifuged for 20 min at 14,000rpm. While centrifuge was running, the wash buffer and elution buffers were made. To bind the protein to the resin, a batch of NI-NTA resin/buffer mix was added. Waste, wash and elution 1 and 2 were collected. The Ni-NTA was stripped with NaOH and water. During the Nanodrop spectrophotometer portion, absorbance of Elution 1 was recorded and then converted into mg/ml. However, the maximum absorption wavelength was 574 nm. On the 3rd part of the labs, samples 1-6 were obtained and centrifuged to extract the pellet. Then, a precast gel was obtained, inserted into the cassette and cleared each lane with TGS buffer from the tank. Then 7ul of Fermentas SM0671 was added to each well.
:
Figure 1a. Culture plate containing ampicillin, bacterial organism BL21 (DE3), and plasmid vector pGEM-gbr22. The bacterial colonies appear a bright purple color because the inserted plasmid encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef. The culture plate demonstrates that transformation of the E. coli cells was successful because the cells are expressing the purple protein.
Figure 1b.
Culture plate containing ampicillin and bacterial organism BL21 (DE3). Since this plate had bacteria without a vector containing resistance to ampicillin, no growth is seen. This plate serves as a control to the plate with the bacteria containing the vector.
Figure 2. Flask containing E. colibacterial culture that was grown over night in a shaking incubator. The culture contains LB, Amp, BL21 (DE3), and pGEM-gbr22. Since the culture is purple, the cells are ready to be harvested.
Figure 3: E. Coli BL21 bacteria that expresses pGEM-gbr22 spun down using Allegra X-15 benchtop centrifuge into a pellet form. The supernatant was removed from the conical tube. Pellets weighed 0.24g (Top) and 0.26g (Bottom). N=2.
"Figure 4: A 50 ml conical tube which contained cell pellet number 2, weighing 0.62 grams, that was formed after spinning the cells down in the large benchtop centrifuge for ~10 minutes centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius."
Figure 5: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.122 N=2
Figure 6: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.129 N=2
Figure 7: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.877. N=2
Figure 8: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.867. N=2
Absorption measured at 0.126 at the wavelength of 574 nm
Beer's Law Calculation
>Determining Concentration of Protein using 280nm
Molecular weight: 25794.2 g/mol
Extinction Coefficient: 38850 M^-1*CM^-1
A=Ebc
A=(.896+.869)/2 =.8825
.8825= c (1) (38850 M^-1*CM^-1)
c=.00002273 mol/L
>Determining Concentration of Protein using Maximal Wavelength
A= (.136+.126)/2 = .131
.131 = c (1) (118,300 M^-1*CM^-1)
c= .00000111 mol/L
Yields from Nanodrop Spectrophotometry
>280nm wavelength: .00002775 mg
(.0002272mol/L)(25794.2g/mol)= .00000555 g/L
.00000555 mg/ml (5ml)= .00002775 mg
>Maximal wavelength: .057926312 mg
(.00000111mol/L)(25794.2g/mol)= .02863156 g/L
(.0286315mg/mL) (5mL) = .05792312 mg
Figure 4: Result of Gel Electrophoresis Using mini-PROTEAN of Various Samples taken during Purification Step for gbr22
Figure 10:Image of Ladder for PageRuler/ Fermentas of MW (kDa) for reference Results/Discussion
In the 3 Bacterial Protein labs, I learned how to transform bacterial cells with a DNA plasmid and grow a started culture of bacteria (purple gbr22 protein). I was able to over express a recombinant protein in bacteria. In these labs I was able to get rid of the cell walls and release the proteins needed to carry on. In the Gel electrophoresis, Ni-NTA affinity helped analyze the samples and estimate the molecular weight, purity and yield of the bacterial gbr11 protein. E. Coli BL21 (DE3) was used as a host for the expression of pGEM-gbr22. The bacteria that didn’t contain the pGEM-gbr22 were killed when it was treated with ampicillin. The bacteria, after incubated with ampicillin, were then incubated in an Erlenmeyer flask for another 24 hours. From this, Sample 1 was collected, and is comprised of E. Coli that contains pGEM-gbr22, LB, and ampicillin. Allegra X-15 centrifuge was then used to spin down the bacteria, followed by the removal of the supernatant. Lysozyme is an enzyme that was used to break down the bacterial cell walls to improve protein or nucleic acid extraction efficiency. Benzonase/Cyanase is a nuclease/enzyme that was used to reduce the viscosity by digesting the DNA/RNA in the mixture. The sample was centrifuged to separate the debris and protein from each other to create sample 2. The supernatant was then filtered, followed by the addition of Ni-NTA resin. Sample 2 consisted of 50 ul of the lysate which contained the Benzonase enzyme. Sample 3 consists of 50 ul of the waste solution that was collected from the Ni-NTA purification. Sample 4 had 50 ul of the flow from the wash that was kept from the Ni-NTA purification. The Wash solution contained 10 ml of final concentration 1x PBS with 20 mM imidazole. Sample 5 contained 50 ul of Elution 1. Elution 1 consisted of 10 ml of final concentration1x PBS with 250 mM imidazole. Sample 6 contained Elution 2. The HIS system is most commonly used protein fusion tag. It is when six histidine residues are attached to the C-terminus, which can be easily used to separate the protein from other cellular proteins. Then, the elution #1 shown in Figure 4 was used for Nanodrop spectrophotometer in order to find the absorbance at 280nm and maximal wavelength. After obtaining the extinction coefficient from external sources, the concentrations of the protein were calculated through Beer’s Law. Moreover, the amount of purified protein in mg was calculated at 280nm and maximal wavelength by knowing volume and concentration of the purified protein. Using the dried gel and protein ladder of Molecular Weight standard, the purified protein was estimated to be 28 kDa. As shown in samples 2-4, the presence of an extra band represented contamination. However, multiple filtrations between the samples decreased the intensity of the extra band with the progression of the lab. In the end, sample 5 contained closely purified gbr22 protein.
Conclusion
The purpose of the lab was to overexpress a recombinant protein in E. coli, to purify gbr22 protein through many filtration methods, and to analyze the samples through gel electrophoresis and Nanodrop spectrophotometer. Verified through the gel, the gbr22 protein was closely purified with little interference from extra cellular materials in sample 5. The techniques gained throughout the lab would frequently be used in independent research when a specific protein needs to be isolated to bind with a top ranking ligands from GOLD. This lab will help in VDS by searching drugs that could inhibit the purple protein that is extracted from bacteria and also to be able extract protein from other various bacterial cells in humans or animals.
Reference [1]. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
[2] European Molecule Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14, 2012)
The pGem-gbr22 protein was expressed, purified and characterized. Recombinant proteins for expression in E. coli is used in biomedical science and E. coli are used as an expression host for proteins from higher organisms to distinguish between the protein and everything else[1]. More specifically, BL21(DE3) is an excellent host strain with deficiency in both lon and ompT proteases, which can enhance accumulation by reducing proteolytic degradation. [2]. The main idea of this lab of expression, purification and characterization, was to transform competent cells and grow cultures to express the colored (purple) protein and harvest cells by using the centrifuge. Another reason was to express and analyze purified gbr22 protein initially grown in bacteria using gel electrophoresis and UV- Vis spectroscopy. After lysing the cell, centrifuging, and using Ni-NTA affinity purification, the gbr22 protein should only remain in the sample.
Materials and methods
Appropriate safety precautions such as gloves, glasses and lab coats to protect against stains, bacteria and chemicals were taken. Also, understanding the labs before beginning them was also a safety precaution. In order to start the first of the three procedures, cloning of the gene and inserting the gene into an expression plasmid was the first steps in expressing the protein. To begin, 25ul of the bacteria was added to each of the two transformation tubes and spun down using a mini centrifuge. 1-2ul of plasmid, which was pGEM-gbr22, was gently added to the bacterial in the DNA tube only and placed it on ice to prepare for the heat shock. After the shock, the samples were placed in ice for another two minutes. 200ul of SOC was added and placed in the incubator. Sterile colirollers were added to each DNA and Control plate to allow bacteria growth on the agar amp plates. The next day, ampicillin was added to the starter culture. A few colonies of the bacteria grown overnight were added to the LB. A few hours later, LB and ampicillin were added to a flask along with BL21(DE2) and then left on the shaking incubator. 8 hours later, the starter culture was transferred to a 125ml flask and put in the shaking incubator for 24 hours. On day three, the bacteria was placed in conical tubes and centrifuged for 10 minutes at 5,000 rpm at 4C. The pellet in the bottom was saved. 1xPBS was added to the cell pellet and vortexed in order to resuspend the cells in Phosphate buffered saline followed by lysozyme. Lysate from above procedure was distributed into several micro centrifuge tubes and centrifuged for 20 min at 14,000rpm. While centrifuge was running, the wash buffer and elution buffers were made. To bind the protein to the resin, a batch of NI-NTA resin/buffer mix was added. Waste, wash and elution 1 and 2 were collected. The Ni-NTA was stripped with NaOH and water. During the Nanodrop spectrophotometer portion, absorbance of Elution 1 was recorded and then converted into mg/ml. However, the maximum absorption wavelength was 574 nm. On the 3rd part of the labs, samples 1-6 were obtained and centrifuged to extract the pellet. Then, a precast gel was obtained, inserted into the cassette and cleared each lane with TGS buffer from the tank. Then 7ul of Fermentas SM0671 was added to each well.
:
Figure 1a. Culture plate containing ampicillin, bacterial organism BL21 (DE3), and plasmid vector pGEM-gbr22. The bacterial colonies appear a bright purple color because the inserted plasmid encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef. The culture plate demonstrates that transformation of the E. coli cells was successful because the cells are expressing the purple protein.
Figure 1b.
Culture plate containing ampicillin and bacterial organism BL21 (DE3). Since this plate had bacteria without a vector containing resistance to ampicillin, no growth is seen. This plate serves as a control to the plate with the bacteria containing the vector.
Figure 2. Flask containing E. colibacterial culture that was grown over night in a shaking incubator. The culture contains LB, Amp,
BL21 (DE3), and pGEM-gbr22. Since the culture is purple, the cells are ready to be harvested.
Figure 3: E. Coli BL21 bacteria that expresses pGEM-gbr22 spun down using Allegra X-15 benchtop centrifuge into a pellet form. The supernatant was removed from the conical tube. Pellets weighed 0.24g (Top) and 0.26g (Bottom). N=2.
"Figure 4: A 50 ml conical tube which contained cell pellet number 2, weighing 0.62 grams, that was formed after spinning the cells down in the large benchtop centrifuge for ~10 minutes centrifuged for 10 minutes at 5,000 rpm at 4 degrees Celsius."
Figure 5: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.122 N=2
Figure 6: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 574 nm and the maximum absorbency level was at 0.129 N=2
Figure 7: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.877. N=2
Figure 8: The Nano-drop spectrophometer was used to measure the Absorbance (10mm) vs. Wavelength (nm) of Elution 1. The maximum wavelength was at 280 nm and the maximum absorbency level was at 0.867. N=2
Absorption measured at 0.126 at the wavelength of 574 nm
Beer's Law Calculation
>Determining Concentration of Protein using 280nm
Molecular weight: 25794.2 g/mol
Extinction Coefficient: 38850 M^-1*CM^-1
A=Ebc
A=(.896+.869)/2 =.8825
.8825= c (1) (38850 M^-1*CM^-1)
c=.00002273 mol/L
>Determining Concentration of Protein using Maximal Wavelength
A= (.136+.126)/2 = .131
.131 = c (1) (118,300 M^-1*CM^-1)
c= .00000111 mol/L
Yields from Nanodrop Spectrophotometry
>280nm wavelength: .00002775 mg
(.0002272mol/L)(25794.2g/mol)= .00000555 g/L
.00000555 mg/ml (5ml)= .00002775 mg
>Maximal wavelength: .057926312 mg
(.00000111mol/L)(25794.2g/mol)= .02863156 g/L
(.0286315mg/mL) (5mL) = .05792312 mg
Figure 4: Result of Gel Electrophoresis Using mini-PROTEAN of Various Samples taken during Purification Step for gbr22
Figure 10:Image of Ladder for PageRuler/ Fermentas of MW (kDa) for reference
Results/Discussion
In the 3 Bacterial Protein labs, I learned how to transform bacterial cells with a DNA plasmid and grow a started culture of bacteria (purple gbr22 protein). I was able to over express a recombinant protein in bacteria. In these labs I was able to get rid of the cell walls and release the proteins needed to carry on. In the Gel electrophoresis, Ni-NTA affinity helped analyze the samples and estimate the molecular weight, purity and yield of the bacterial gbr11 protein. E. Coli BL21 (DE3) was used as a host for the expression of pGEM-gbr22. The bacteria that didn’t contain the pGEM-gbr22 were killed when it was treated with ampicillin. The bacteria, after incubated with ampicillin, were then incubated in an Erlenmeyer flask for another 24 hours. From this, Sample 1 was collected, and is comprised of E. Coli that contains pGEM-gbr22, LB, and ampicillin. Allegra X-15 centrifuge was then used to spin down the bacteria, followed by the removal of the supernatant. Lysozyme is an enzyme that was used to break down the bacterial cell walls to improve protein or nucleic acid extraction efficiency. Benzonase/Cyanase is a nuclease/enzyme that was used to reduce the viscosity by digesting the DNA/RNA in the mixture. The sample was centrifuged to separate the debris and protein from each other to create sample 2. The supernatant was then filtered, followed by the addition of Ni-NTA resin. Sample 2 consisted of 50 ul of the lysate which contained the Benzonase enzyme. Sample 3 consists of 50 ul of the waste solution that was collected from the Ni-NTA purification. Sample 4 had 50 ul of the flow from the wash that was kept from the Ni-NTA purification. The Wash solution contained 10 ml of final concentration 1x PBS with 20 mM imidazole. Sample 5 contained 50 ul of Elution 1. Elution 1 consisted of 10 ml of final concentration1x PBS with 250 mM imidazole. Sample 6 contained Elution 2. The HIS system is most commonly used protein fusion tag. It is when six histidine residues are attached to the C-terminus, which can be easily used to separate the protein from other cellular proteins. Then, the elution #1 shown in Figure 4 was used for Nanodrop spectrophotometer in order to find the absorbance at 280nm and maximal wavelength. After obtaining the extinction coefficient from external sources, the concentrations of the protein were calculated through Beer’s Law. Moreover, the amount of purified protein in mg was calculated at 280nm and maximal wavelength by knowing volume and concentration of the purified protein. Using the dried gel and protein ladder of Molecular Weight standard, the purified protein was estimated to be 28 kDa. As shown in samples 2-4, the presence of an extra band represented contamination. However, multiple filtrations between the samples decreased the intensity of the extra band with the progression of the lab. In the end, sample 5 contained closely purified gbr22 protein.
Conclusion
The purpose of the lab was to overexpress a recombinant protein in E. coli, to purify gbr22 protein through many filtration methods, and to analyze the samples through gel electrophoresis and Nanodrop spectrophotometer. Verified through the gel, the gbr22 protein was closely purified with little interference from extra cellular materials in sample 5. The techniques gained throughout the lab would frequently be used in independent research when a specific protein needs to be isolated to bind with a top ranking ligands from GOLD. This lab will help in VDS by searching drugs that could inhibit the purple protein that is extracted from bacteria and also to be able extract protein from other various bacterial cells in humans or animals.
Reference
[1]. Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
[2] European Molecule Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 14, 2012)